Extreme Physiological Plasticity in a Hibernating Basoendothermic Mammal, Tenrec Ecaudatus Michael D

Home , Amblysomus, Golden mole, Hemicentetes, Hottentot golden mole, Lesser hedgehog tenrec, Tenrec

RESEARCH ARTICLE Extreme physiological plasticity in a hibernating basoendothermic mammal, Tenrec ecaudatus Michael D. Treat1,*, Lori Scholer1, Brandon Barrett1, Artur Khachatryan1, Austin J. McKenna1, Tabitha Reyes1, Alhan Rezazadeh1, Charles F. Ronkon1, Dan Samora1, Jeremy F. Santamaria1, Claudia Silva Rubio1, Evan Sutherland1, Jeffrey Richardson2, John R. B. Lighton2 and Frank van Breukelen1,*

ABSTRACT hypothetical placental ancestor Schrëwdinger. Schrëwdinger was Physiological plasticity allows organisms to respond to diverse presumed to lack a cloaca, but to have a large and gyrencephalic conditions. However, can being too plastic actually be detrimental? brain, zygomatic arches and tympanic bullae, and internal testes ’ Malagasy common tenrecs, Tenrec ecaudatus,havemany (O Leary et al., 2013). In comparison, tenrecs have a cloaca and, in plesiomorphic traits and may represent a basal placental mammal. the case of Tenrec ecaudatus, have the smallest brain relative to We established a laboratory population of T. ecaudatus and found body mass of all extant mammals (including monotremes and extreme plasticity in thermoregulation and metabolism, a novel marsupials), are lissencephalic, lack zygomatic arches and tympanic hibernation form, variable annual timing, and remarkable growth and bullae, and have internal testes that are located near the kidneys (Lillegraven and Eisenberg, 1983). These features would suggest reproductive biology. For instance, tenrec body temperature (Tb)may approximate ambient temperature to as low as 12°C even when that tenrecs may be reminiscent of basal placental mammals. In Lovegrove’s (2012) plesiomorphic–apomorphic endothermy tenrecs are fully active. Conversely, tenrecs can hibernate with Tb of 28°C. During the active season, oxygen consumption may vary 25-fold model, he posited that ancestral mammals were heterothermic and more rigid homeothermy is a derived condition. Lovegrove (2012) with little or no change in Tb. During the austral winter, tenrecs are consistently torpid but the depth of torpor may vary. A righting assay defines a basoendotherm simply as any mammal with a body temperature (Tb) in the 20th percentile of the frequency distribution revealed that Tb contributes to but does not dictate activity status. Homeostatic processes are not always linked, e.g. a hibernating tenrec of the mammalian Tb dataset, meaning they must exhibit resting Tb experienced a ∼34% decrease in heart rate while maintaining constant <35°C. Lovegrove (2012) notes that the endothermic conditions of body temperature and oxygen consumption rates. Tenrec growth rates extant basoendotherms were likely also exhibited by Cretaceous or vary but young may grow ∼40-fold in the 5 weeks until weaning and early Cenozoic mammals. Tenrecs are considered basoendothermic may possess indeterminate growth as adults. Despite all of this because of the presumed phylogenetic placement of afrotherians profound plasticity, tenrecs are surprisingly intolerant of extremes in and because existing data suggest their Tb is generally <35°C ambient temperature (<8 or >34°C). We contend that while plasticity (Lovegrove, 2012). Tb is also moderately variable in many of the may confer numerous energetic advantages in consistently moderate basal mammalian taxa; some monotremes, marsupials, xenarthrans environments, environmental extremes may have limited the success and afrotherians experience active Tb that varies by as much as 8°C and distribution of plastic basal mammals. (Schmidt-Nielsen et al., 1966; McManus, 1969; Scholl, 1974; Gilmore et al., 2000; Oelkrug et al., 2012). The limited existing data KEY WORDS: Thermoregulation, Oxygen consumption, Variable on active tenrecs suggest a Tb always <35°C with moderate variation body temperature (Tb=31.82±0.098°C, mean±s.e.m.; Racey and Stephenson, 1996; Poppitt et al., 1994; Lovegrove et al., 2014, Levesque and INTRODUCTION Lovegrove, 2014; Levesque et al., 2014). Tenrecs are basoendothermic placental mammals primarily from More pronounced heterothermy in small mammals occurs during Madagascar that are phylogenetically placed in Afrotheria hibernation in conjunction with metabolic depression. Importantly, (Lovegrove, 2012; Everson et al., 2016). Prevailing thought is that metabolic depression and low Tb are linked, e.g. it is thought that a small tenrec species arrived in Madagascar 30–56 MYA and the metabolic depression allows Tb to decrease and that the low Tb radiated into the remaining extant species (Poux et al., 2008; may allow depression of metabolic processes (e.g. van Breukelen and Everson et al., 2016). This tremendous adaptive radiation resulted in Martin, 2015). For instance, hibernating ground squirrels experience species that are terrestrial, fossorial, arboreal and even aquatic decreases of Tb to as low as −2.9°C (Barnes, 1989) and oxygen (Garbutt, 1999). Tenrecs have many ‘ancestral’ features that seem consumption rates to as low as 1/100th of active rates. Hibernation even more plesiomorphic than those of the reconstructed is also found in basal mammals including the echidna, some marsupials, and afrotherians like the elephant shrew, golden moles and tenrecs (Ruf and Geiser, 2015; Scantlebury et al., 2008). All five 1School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV 89154, spiny tenrec species, including the focus of this work, T. ecaudatus, USA. 2Sable Systems International, Las Vegas, NV 89032, USA. are known to hibernate (Stephenson and Racey, 1994). The limited *Authors for correspondence ([email protected]; data on hibernating T. ecaudatus demonstrate that Tb follows soil [email protected]) temperature for the duration of the hibernation season with no evidence of brief periods of euthermy between bouts of torpor M.D.T., 0000-0002-3247-4601; L.S., 0000-0002-7596-2058; T.R., 0000-0001- 7128-6366; D.S., 0000-0001-7452-8922; F.v.B., 0000-0001-9653-0273 (Lovegrove et al., 2014). The lack of interbout arousals is unique for small hibernators. Interestingly, dwarf lemurs (Cheirogaleus medius)

Received 6 June 2018; Accepted 20 August 2018 will arouse from hibernation if they experience significant periods Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb185900. doi:10.1242/jeb.185900 wherein ambient temperature (T ) is <30°C (Dausmann et al., 2004; Respirometry a ̇ Dausmann et al., 2005). Soil temperature in the T. ecaudatus field Aerobic respirometry (oxygen consumption, VO2, and carbon ∼ ̇ study was as low as 22°C for several months. An interesting dioxide production, VCO2) was measured with the Promethion question is whether T. ecaudatus would arouse if hibernating at an Continuous Metabolic Phenotyping System and ExpeData even lower Ta or whether these tenrecs simply hibernate continuously. software (Sable Systems International, Las Vegas, NV, USA). The If tenrecs are indeed plesiomorphic, perhaps a lack of interbout Promethion system auto-baselines for 30 s every 15 min with arousal was an ancestral hibernation characteristic. If so, what may be environmental air, otherwise sampling from each metabolic gleaned from characterizing the physiology of tenrec hibernation? chamber every second. Metabolic chambers were constructed from We established a breeding colony of T. ecaudatus in order to Lettuce and Produce Keeper containers (∼1.8 l; Container Store, examine key parameters affecting their life history in the controlled Coppell, TX, USA). Flow rates were 500 ml min−1. Preliminary setting of the laboratory. We found captive tenrecs to exhibit analyses revealed tenrecs to display three physiological states: extreme physiological plasticity. Active and hibernating tenrecs hibernation, facultative torpor and active. During the austral winter, may have identical Tb. Numerous other tenrec life history traits are tenrecs were hibernating and showed the typical signs of torpor, e.g. also highly plastic. We question why such a plastic mammal is not Tb approached Ta and tenrecs were lethargic even when disturbed. more successful, i.e. if being plastic has selective advantages, then We noted that upon entry into the metabolic chamber, Tb would why do the tenrecs not have a more robust global distribution? We sometimes slowly increase by 1–6°C for various periods (taking up provide a surprising explanation that being too plastic may have to 3–4 h to peak with a similar period to return to the original value) limited the success and distribution of such mammals in more in what appeared to be a partial thermal arousal. Importantly, these variable environments. Moreover, by being more precise in their tenrecs maintained a lethargic state typical of torpor and never thermoregulation and physiology, the more modern boreoeutherian aroused to an active state. Furthermore, at Ta <20°C, tenrecs were mammals may have evolved to persist in more variable noted to experience 30–45 min apneic periods. To avoid sampling environments, leading to a wider global distribution. bias caused by the partial thermal arousals and the apneic periods, data for hibernating tenrecs represent an 8 h period of stable Tb (after MATERIALS AND METHODS ∼14 h of a typical 24 h assay). During the active season (after the Acquisition and maintenance of the T. ecaudatus colony resumption of feeding, i.e. October to April), tenrecs were also Forty wild-caught common tenrecs, T. ecaudatus (Schreber 1778), sometimes facultatively torpid wherein Tb approached Ta and were imported under federal and state permit from Mauritius in June animals displayed lethargy typical of torpor. Care was taken to 2014. Approval for this study was granted by the University of gently place the tenrecs into the metabolic chamber as these animals Nevada, Las Vegas Institutional Animal Care and Use Committee. were prone to disturbance and would occasionally arouse from the Tenrecs were typically maintained on Mazuri insectivore diet (Saint facultative torpor into an active state (in contrast to hibernators, Paul, MN, USA). Tenrecs were supplemented with Purina Puppy which remained consistently lethargic). Analyses were restricted to Chow (St Louis, MO, USA) and/or Hills A/D diet (Topeka, KS, 90 min for these assays because of the lability of facultative torpor. USA). Tenrecs were weighed regularly to monitor health. All Active-season tenrecs that demonstrated no signs of lethargy had ̇ tenrecs were maintained on a 12 h:12 h light:dark cycle. For remarkably variable resting VO2 and Tb. To best illustrate this temperature-controlled experiments, tenrecs were individually variability, the analyses comprised a 60 min period with either the ̇ housed in rat cages and/or metabolic chambers within an highest or lowest stable VO2. We defined stable as a period where environmental chamber that maintained precise temperature control. there was little variability that may have indicated obvious activity or movement. The chamber was small enough to prevent the animals

Measurement and validation of Tb from exercising. Each analysis group consisted of eight tenrecs Tenrecs arrived in a torpid state during their hibernation season except for facultative torpor at 28°C, where only seven tenrecs were ̇ −1 (austral winter), which precluded surgery to implant temperature available for the analysis. All VO2 data are presented as ml O2 g −1 loggers. However, we found that Tb could be reliably and accurately body mass h . Box plots were generated using standard box plot determined through the use of pre-calibrated iButton temperature parameters wherein the box identifies the first quartile (bottom line), loggers (DS1922L; Maxim Inc., San Jose, CA, USA) held against the median (middle line) and third quartile (top line) of the dataset; the chest by specially designed harnesses. The iButtons were typically set whiskers represent the maximum and minimum values for each for high-resolution polling (∼4000 samples) and sampling frequency dataset. In order to best demonstrate the extreme variability in typically ranged from 1 to 10 min depending on the desired polling T. ecaudatus aerobic metabolism, we chose to omit outlier analyses duration. iButton data were downloaded at the end of each experiment and all data are included. period. In an unrelated study, we collected tissue samples from active and torpid tenrecs that were harnessed with chest iButtons at the Electrocardiogram (ECG) measurement and heart rate time of death. We found notable regional heterothermy within the analysis abdomen with as much as ∼3°C variation in temperature depending ECGs were collected through use of a three-lead ECG module, on probe location (e.g. lower abdomen versus near the liver). In order iWorx Data Recorder and LabScribe (IWX-214 and LabScribe v3; −1 to allow consistent Tb measurement, we measured Tb at the liver with IWorx Systems Inc., Dover, NH, USA). Heart rate (beats min ) a thermocouple. When tenrecs were housed at a Ta of 12, 20 or 28°C, was determined for a minimum of three consecutive minutes every liver temperature ranged from 12.5 to 30.5°C. When compared with 10 min of the analysis period. the matching chest temperatures, liver temperature was 0.467±0.18°C (mean±s.e.m.) higher (P<0.05, paired t-test, N=21; r2>0.99 in Righting response time (RRT) assay Fig. S1A). These results may be expected as liver temperature in other Tenrecs (N=8, except for the April assay at 28°C, wherein 7 tenrecs mammals is slightly higher than other indicators of core Tb were used because of an unexpected mid-trial death) were housed at (Dewasmes et al., 2003). Nevertheless, T. ecaudatus chest 12, 20 or 28°C for a minimum of 24 h prior to the righting trials. To temperature reliably and accurately reflects Tb. avoid potential bias from disturbance, the order in which animals Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb185900. doi:10.1242/jeb.185900 were used for each trial was randomized through use of a random A number generator. We found no correlation between this order and 35 RRT. Each trial followed strict timing protocols throughout the year, e.g. animals were placed into the apparatus within 20 s of removal 30 from the environmental chamber and there were 3 min intervals between animals. Animals were placed on their backs in a cradle and allowed 60 s to attempt to right themselves. Righting was video 25 recorded and the time to right was determined from the video (Movies 1 and 2). 20

Emergence analysis 15 Most hibernators experience marked changes in Tb at the cessation of the hibernation season as active behaviors resume. However, 10 even active tenrecs have variable Tb. As a result, we used body mass 0 122436 changes as tenrecs resumed eating following emergence from hibernation to estimate date of emergence. B 35 Statistics As appropriate, data were analyzed using ANOVA with subsequent Fisher’s least significant difference (LSD) post hoc analyses or 30 paired and unpaired Student’s t-test. Statistical differences were assumed when P<0.05. A Bonferroni adjustment in Fig. 2 was used 25 because of the large number of comparisons. All data presented with (°C)

̇ b error calculations (e.g. VO2, mass analysis, etc.) are given as means T 20 ±s.e.m. unless otherwise noted.

15 RESULTS

Tb 10 Tb in captive tenrecs was extremely plastic during both hibernation 0122436 and the active season (Fig. 1). During the austral winter, tenrecs can seasonally hibernate at low or high Ta from at least 12 to 28°C C (Fig. 1). During hibernation, tenrec T approximates T and tenrecs b a 35 are consistently lethargic. Importantly, when housed at 12°C and disturbed, Tb of hibernating tenrecs was noted to increase only 30 ∼1–6°C. Remarkably, active tenrecs may also experience Tb ranging from ∼12 to 34°C that frequently waxes and wanes throughout the day. Even with a Tb of ∼12.5°C, these animals 25 display no lethargy or ataxia and are capable of running and swimming. Although active tenrec Tb is oftentimes quite variable 20 throughout the day, Tb may also be relatively stable for shorter periods of time (Fig. S1B–D). Not surprisingly, the differences 15 between Tb and Ta decreased as Ta increased, e.g. animals maintained at a Ta of 28°C had less variation in Tb than animals 10 maintained at a Ta of 12°C. Active-season tenrecs were also capable 0 122436 of using torpor facultatively at a Ta of 12, 20 and 28°C (Fig. 1). Time (h) Active-season tenrecs that had resumed active behaviors, e.g. eating T Tenrec ecaudatus and reproduction, were considered to be in facultative torpor when Fig. 1. Extreme variability of body temperature ( b)of . Tenrecs were housed at an ambient temperature (T ) of (A) 12°C, (B) 20°C or the tenrec became lethargic and Tb approximated Ta for several days. a In contrast to hibernating animals, facultatively torpid tenrecs were (C) 28°C. Note the lack of homeothermy in active tenrecs (red, green and orange circles). Individual active tenrecs may have low or high Tb that can sensitive to disturbance wherein animals would arouse into a fully change >2.5°C h−1. Alternatively, active-season tenrecs may spontaneously active and alert state with no indication of lethargy or ataxia. enter periods of facultative torpor (purple squares) where Tb approximates Ta. During the austral winter, tenrecs maintain a constant state of hibernation (blue Metabolism squares). Metabolism in active tenrecs may vary by 25-fold (Fig. 2). In order to best demonstrate this extreme variability, 60 min periods of the despite these active tenrecs having higher Tb (ANOVA, P>0.05). ̇ ∼ −1 −1 highest (active high) and lowest (active low) stable respirometry Tenrecs may also have similar high VO2 ( 1mlO2 g h ) and Tb data are presented in Fig. 2. Oxygen consumption rate in active (∼28°C) even when there is an 8°C difference in Ta (Ta of 12 or tenrecs did not simply switch between high and low levels, but 20°C; Fig. 2A,B). Additional examples of how metabolism may be rather occurred along a continuum of rates between the active high disconnected from T in individual tenrecs are provided in Figs S2 ̇ b and active low levels. Tenrec VO2 was only partially dependent on and S3. T . For instance, the active low V̇ in Fig. 2A was not statistically When all data were analyzed together, no differences in V̇ were b ̇ O2 O2 Journal of Experimental Biology different from the VO2 of facultative torpid or hibernating tenrecs found between the hibernating, facultative torpid or even the active

3 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb185900. doi:10.1242/jeb.185900

A B C 1.1 1.4 2 1 1.8 1.2 0.9 28.0±3.4 1.6 2 O )

· 0.8 V 1 1.4 –1 28.4±3.6

h 0.7 1.2 –1 0.6 0.8 g

2 1 0.5 0.6 0.4 0.8 (ml O Mass-specific 0.3 0.4 28.0±3.3 0.6 21.3±0.5 0.2 0.4 31.7±1.1 0.2 32.0±0.8 0.1 18.1±7.0 13.0±0.7 29.0±0.4 29.3±0.4 12.7±0.6 20.7±0.1 0.2 0 0 0 Active Active Facultative Hibernating Active Active Facultative Hibernating Active Active Facultative Hibernating high low torpor high low torpor high low torpor ̇ Fig. 2. Plastic oxygen consumption (VO ) in tenrecs. Ta was (A) 12°C, (B) 20°C or (C) 28°C. Data represent box plots of the highest (active high) or lowest ̇ 2 (active low) VO2 in non-torpid active-season tenrecs (see Materials and Methods, N=8). For comparison, data from active-season facultatively torpid tenrecs and hibernating tenrecs are included. Note, aerobic metabolism is not necessarily related to Tb or Tb−Ta differentials, e.g. active high values for 12 and 20°C are ̇ similar. All active high groups showed significantly greater VO2 than all other comparisons. No other differences were detected when all groups were analyzed together (P<0.05; ANOVA). Numbers associated with the boxes represents Tb (°C; means±s.d.). low state at Ta of 12, 20 or 28°C (Fig. 2; ANOVA, P>0.05). Only Righting response when active animals were removed from the analyses (i.e. reducing As indicated above and unlike in other hibernators, we observed that the number of comparisons by comparing only facultative torpid and Tb was a poor indicator of metabolic status. We noted that hibernating hibernating tenrecs) did statistical differences between temperatures tenrecs, unlike common hibernator models such as ground squirrels, ̇ become evident; in these comparisons, VO2 decreased with decreasing were not simply torpid or non-torpid during the hibernation season. temperature as expected (Q10=3.7 for hibernating tenrecs and 2.0 for Rather, early season hibernating tenrecs typically appeared ‘less’ facultatively torpid tenrecs between 12 and 28°C; ANOVA, P<0.05). torpid and more responsive to disturbance, mid-season hibernators Both active and torpid tenrecs experienced extended periods were deeply torpid and resisted disturbance, and late season wherein there were unexpected relationships between T , V̇ and hibernators were less torpid. Furthermore, we found that T also ̇ b O2 b heart rate. For instance, identical VO2 values were accommodated by partially predicted hibernation depth, e.g. a cold hibernating tenrec heart rates that varied by 34% (Fig. 3). Additional examples of was more likely to be profoundly lethargic and a warm tenrec was metabolic flexibility are provided in Figs S2 and S3. more likely to be partially ambulatory and less ataxic when handled. Importantly, hibernating tenrecs were always somewhat lethargic and ataxic regardless of Ta. In order to best illustrate annual torpor use and this concept of ‘more or less’ torpid, we monitored lethargy in T. ecaudatus throughout the year through the use of a righting 1 response assay (Fig. 4; Movies 1 and 2). In general, increased )

–1 lethargy occurred when animals were housed at a lower Ta,inboth 2

min active and hibernation seasons. Tenrecs housed at a Ta of 12°C were 2 CO · V usually less likely to right themselves within the 60 s assay while

CO 0.5 or 2

or tenrecs housed at a Ta of 28°C rarely spent more than a few seconds O · 2 V righting themselves. Hibernating tenrecs at a Ta of 28°C were very readily ‘aroused’ and although they were able to right quickly, we still (ml O 0 35 noted some ataxia in these animals. Interestingly, some animals with

) Tb near Ta to as low as 12°C were able to right themselves quickly. –1 Conversely, some warm animals took the full 60 s to right (Fig. S4). At a Ta of 12°C, a defined seasonality was present wherein

25 (°C) b T. ecaudatus appeared most lethargic in June–July and most active T

(beats min in December, consistent with our observations of the hibernation and H f active seasons (Fig. 4A). 15

0 123 4 567 8 Emergence from hibernation Unlike in other hibernators, the end of the hibernation season in Time (h) tenrecs was a gradual transition and occurred over a couple of Fig. 3. Demonstration of the lack of coordination of basic homeostatic weeks. As individual tenrecs transitioned out of hibernation, they mechanisms. T was 16°C. Torpid V̇ was the same in the two highlighted a O2 slowly became less lethargic, less ataxic and more aware of their −1 (red and blue) 60 min periods (0.171 ml O2 min ). However, while heart rate −1 surroundings (L.S., M.D.T. and F.v.B., personal observations). ( fH) in the red phase averaged 28.8±2.0 beats min , heart rate during − Tenrecs then resumed active-season behaviors like eating, growth the blue phase averaged 19.0±1.3 beats min 1, representing a ∼34% ̇ – and reproduction. By tracking food intake and body mass, decrease in heart rate despite identical VO2. Note, the 30 45 min periods between breaths in this hibernating tenrec. approximate dates of emergence from hibernation for each tenrec Journal of Experimental Biology

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A were determined (Fig. 5). Some tenrecs emerged as early as August 0 or as late as January, with most tenrecs having exited hibernation by October or November (Fig. S5A).

10 Growth rates To establish our colony, we obtained wild-caught juvenile and adult 20 tenrecs that were generally small (262±15.9 g; N=35). Upon emergence from hibernation, these tenrecs ate copious amounts of food and grew tremendously in a short period of time (2.5- to 3-fold 30 in ∼4 months; average maximal daily mass gain of 44.6± 4.0 g day−1; N=35; Fig. 5). This growth was not simply the result 40 of fat accumulation as these animals also grew in scale. Furthermore, dramatic growth was not limited to juveniles as 50 adult tenrecs also experienced similar growth (Fig. S5B). Despite a birth mass of 12.15±0.36 g (N=31; four litters), captive tenrecs oftentimes weigh >400 g by the time they wean at 60 5 weeks. We observed juvenile tenrecs eating from their mothers’ Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. mouths and/or eating soft foods during the first week post-partum. The profound growth is not simply a function of tenrecs growing as B quickly as possible as there may be surprising consistency of 0 growth rates within a given litter (Fig. S5C,D). Further, there may be multiple growth rate trajectories represented within a given 10 litter, e.g. some tenrecs may grow quickly while others may grow more slowly. Although most of our tenrecs grew quickly prior to the hibernation season, some juveniles entered hibernation at a 20 body mass less than 150 g. These smaller tenrecs experienced the same profound post-hibernation growth as our wild-caught 30 tenrecs. RRT (s) RRT 40 Tb in pregnant and post-partum tenrecs Prior to parturition, tenrec Tb may be variable during gestation (Fig. 6; Fig. S6). Following parturition, females experienced 50 increased and more stable Tb (above 30°C; Fig. 6; Fig. S6). In one extraordinary case, a pregnant female housed at a Ta of 12°C 60 experienced a >24 h period wherein Tb was 13.5±0.5°C (Fig. S6G). We estimated that she was ∼3 weeks into her pregnancy. Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan. C 0

1200 10

1000 20

30 800

40 600

50 Body mass (g) 400

60 200

Feb. Mar. Apr. May Jun. Jul. Aug. Sep. Oct. Nov. Dec. Jan.

Calendar month 0 Sep. Oct. Nov. Dec. Jan. Fig. 4. Demonstration of seasonal and Ta effects on righting response time (RRT). Tenrecs were housed at a Ta of (A) 12°C, (B) 20°C or (C) 28°C. Fig. 5. Tenrecs may experience extreme growth following exit from Filled circles of different colors represent individual tenrecs. Squares represent hibernation. Tenrecs were observed to gradually increase in activity for as means±s.e.m. of that month’s RRT. Seasonality was more evident in tenrecs much as 2 weeks prior to the onset of very rapid growth and mass gain. housed at 12°C than at 28°C; however, even when housed at 28°C, some Different colors represent individual tenrecs. Average daily maximum mass − animals displayed a RRT of >20 s. gain was 44.6±4 g day 1, N=35. Journal of Experimental Biology

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−1 −1 A (active low) or as high as 0.9916±0.052 ml O2 g h (Fig. 2). 36 Surprisingly, T did not dictate metabolism. Tenrecs housed at 12 b ̇ ̇ and 20°C maintained similar VO2 and Tb, e.g. active high VO2 across 34 a 60 min period for animals maintained at a Ta of 20°C was 1.0856± −1 −1 0.097 ml O2 g h despite these animals having the same Tb ∼ 32 ( 28°C) as the active high animals that were maintained at 12°C. Remarkably, Tb of the active low animals housed at 20°C was also ∼28°C, despite these tenrecs having a ∼6.4-fold lower V̇ than that 30 O2 of the active high animals. We note that active low metabolism is not simply torpor use. Tb was higher than Ta in active low tenrecs. 28 Furthermore, active low tenrecs did not exhibit the lethargy and ataxia of facultatively torpid tenrecs found during the active season. 26 Although the data presented here do not offer a mechanistic basis, it appears tenrecs may be able to modulate heat retention and 24 04191023 5678 conductance independent of other physiological parameters like B V̇. An alternative explanation for the lack of a relationship between (°C) O2 b 36 ̇ T VO2 and Tb is that there could be a significant anaerobic component to metabolism. The data for V̇ generally coincide with those for 34 CO2 V̇ and do not support the use of anaerobic metabolism. Further, O2 ̇ identical Tb and VO2 values could be accommodated by heart rates 32 that vary by 34% (Fig. 3). These data support a notion that metabolism in tenrecs may simply be plastic in comparison to Tb.In 30 common tenrecs, the large variation in metabolism is not associated with the use of torpor or hibernation. 28 In most mammalian species, such profound changes in Tb and metabolism would be associated with the use of torpor or 26 hibernation (Ruf and Geiser, 2015; van Breukelen and Martin, 2015). Torpor is traditionally defined as a reduction in metabolism 24 to levels below what is required to maintain normal metabolic 012 34567 activities. In many cases, torpid Tb may approach Ta and is Time (days) associated with extreme metabolic depression. In other cases, a Fig. 6. Tenrecs can invoke periods of higher endothermy/homeothermy. more moderate depression of metabolism results in torpid Tb being

(A) A non-pregnant tenrec shows typical variation in Tb. (B) Tenrecs that give higher than Ta. The depressed metabolism serves as a mechanism to birth (shaded area indicates day of parturition) maintain a higher and less conserve energy during times of limited resource availability or variable Tb post-partum. These tenrecs were housed at room temperature. environmental stress. Active suppression of metabolism has been previously demonstrated in hibernating black bears (Tøien et al., DISCUSSION 2011). Upon exit from hibernation, bear Tb increases by ∼2°C. ̇ – Common tenrecs have highly variable Tb and rates of oxygen However, VO2 slowly increases over the ensuing 2 3 weeks. In consumption (Figs 1–3 and 6; Figs S1–S4 and S6). This variability tenrecs, remarkable metabolic suppression is seen during facultative ̇ is not restricted to the hibernation season in tenrecs. Moderately torpor and hibernation. For instance, hibernating tenrec VO2 at 12°C −1 −1 variable active-season Tb is prevalent in many of the basal is 0.0115±0.023 ml O2 g h . For comparison, hibernating Arctic ̇ −1 −1 mammalian taxa. In monotremes like the echidna Tachyglossus ground squirrel VO2 at 4°C is 0.012 ml O2 g h (Buck and aculeatus, active T was 30.7±1.03°C with a range of as much as Barnes, 2000). Interestingly, euthermic (T ∼36°C) Arctic ground b ̇ b ̇ 4.1°C when held at a Ta between 0 and 25°C (Schmidt-Nielsen squirrel VO2 at a Ta of 2°C approximates our active high VO2 at a Ta of et al., 1966). In a marsupial, the opossum Didelphis viginiana, Tb 12 and 20°C (Karpovich et al., 2009), indicating that tenrecs are not was typically 33–35°C, with the lowest recorded Tb reaching 30.8°C simply hypometabolic. It appears that tenrecs are able to suppress when held at a Ta from −10 to 32°C (McManus, 1969). In a aerobic metabolism to a degree equal to that of the most competent xenarthran, the brown-throated three-toed sloth, Bradypus hibernators despite being at least 8°C warmer. variegatus, active Tb ranged from 28 to 35°C with the circadian In all described mammalian hibernators to date, the hibernation cycle (Gilmore et al., 2000). Of note is that these ranges for Tb are phenotype consists of bouts of torpor interrupted by brief periodic relatively moderate, with few animals experiencing variations of returns to euthermy wherein critical homeostatic processes are more than 8°C. Even in other afrotherians like elephant shrews, Tb restored (van Breukelen and Martin, 2015). Lovegrove et al. (2014) varied by only a few degrees during the active season (Mzilikazi and found that wild T. ecaudatus did not experience these interbout Lovegrove, 2004; Mzilikazi and Lovegrove, 2005; Oelkrug et al., arousals. Common tenrecs hibernate in small, underground burrows 2012). We found ∼20°C variation in Tb in both active and ∼1 m deep that are sealed to the outside with soil for the 8–9 month hibernating tenrecs. hibernation season (Lovegrove et al., 2014; Rand, 1935). Recent In T. ecaudatus, active-season aerobic metabolism (active low) video evidence showed 13 torpid tenrecs were removed from a may be statistically comparable to that of even torpid tenrecs and single hibernaculum by hunters, suggesting tenrecs may hibernate independent of a reduction in Tb (Fig. 2). Conversely, oxygen socially (https://youtu.be/w8OAm_eeNd8). Soil temperatures in the consumption in active tenrecs may also be ∼25-fold higher (active Lovegrove et al. (2014) field study were never <22°C, which ̇ high). For instance, at a Ta of 12°C, stable VO2 across a 60 min precluded colder tenrec Tb. Data for fat-tailed dwarf lemurs, −1 −1 period in active tenrecs was as low as 0.0405±0.021 ml O2 g h Cheirogaleus medius, demonstrate that periodic interbout arousals Journal of Experimental Biology

6 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb185900. doi:10.1242/jeb.185900 occur only if the tree hole is <30°C during its hibernation season higher blood pressure and pulses in kidney function without (Dausmann et al., 2004). An important question is whether lower Tb increases in Tb. We found that tenrecs hibernating at a Ta of 12°C are (i.e. Tb <22°C) during hibernation would elicit the use of periodic able to urinate (M.D.T., L.S. and F.v.B., personal observations). In arousals in common tenrecs (van Breukelen and Martin, 2015). ground squirrels, kidney function is restricted to the interbout When held at a Ta between 12 and 28°C during the hibernation arousal (Jani et al., 2013). Our laboratory is currently investigating season, we found captive tenrecs had a core Tb that closely these ideas. approximated Ta (Fig. 1; Figs S1B–D, S2) and found no evidence of In common models of hibernation like arctic ground squirrels, the natural periodic interbout arousals even at a Tb as low as ∼12°C. timing of immergence and emergence from hibernation is generally Upon disturbance, some tenrecs experienced ∼1–6°C increases in synchronized and highly predictable to within a few days (Sheriff Tb that may last from <1 h to 14 h. We noticed these partial thermal et al., 2011). In T. ecaudatus, the timing seems much more plastic. arousals occurred in close proximity to the daily checks or We noted that tenrecs slowly exited hibernation and became more weighings. Tenrec thermal arousals are highly variable and lack aware of their surroundings and less lethargic over the course of predictability, features inherent to canonical interbout arousals. If ∼2 weeks. Tenrecs then resumed active-season behaviors, e.g. rapid the function of an interbout arousal is to allow re-establishment of and coordinated movement, eating, voiding of waste products, and homeostatic processes with high Tb (van Breukelen and Martin, reproduction. The timing of emergence of our captive tenrec 2001), it is difficult to see how a modest increase in Tb, e.g. 12.5– population varied by ∼5 months (Fig. 5; Fig. S5A). We have 15°C, would accomplish those functions. Further, these same partial maintained the colony since 2014 and have observed no indications thermal arousals occurred at a Ta of 28°C where no similar need that the annual cycle is shifting from a southern towards a northern could be rationalized. As a result, we contend that the periods of hemisphere cycle. Tenrecs immerged into hibernation as early as increased Tb in captive tenrecs do not represent a natural feature of January and in some exceptional cases as late as July (data not tenrec hibernation and should not be considered canonical interbout shown). Most tenrecs were hibernating by May. arousals. Instead, we liken these partial thermal arousals to Plastic growth rate trajectories were evident in neonatal, juvenile unsuccessful alarm arousals. In ground squirrels, handling of and adult tenrecs. While the profound mass gain in neonatal tenrecs torpid animals prematurely induces an interbout arousal where Tb is not as extreme as the ∼25% daily mass gain in neonatal hooded fully returns to euthermic levels for the prescribed 12–20 h period seals, Cystophora cristata, which wean at ∼3–5 days of age, the before continuing the next torpor bout (alarm arousal; Utz and van mass gain in these seals is mostly fat (Bowen et al., 1985), whereas Breukelen, 2013). Importantly, not all manipulations cause alarm in tenrecs the mass gain reflected changes in skeletal dimension. arousals. In some squirrels, Tb may increase ∼1–2°C before Interestingly, profound growth even occurred in adult tenrecs. returning to the pre-handling Tb. In our experience with ground Indeterminate growth is considered the ancestral condition in squirrels, we have never seen a partial arousal where the increase in amniotes (Hariharan et al., 2016); during indeterminate growth, Tb was greater than 2°C. Tenrecs never fully arouse from torpor increases in the length of the major body axis occur after the animal during the hibernation season and there is always some indication of is thought to have reached a mature size. That our wild-caught adult lethargy and ataxia. This continuous lethargy/torpor may be tenrecs grew 2.5±0.37-fold suggests a degree of indeterminate demonstrated by examination of heart rates. During the active growth that appears unique in the mammalian world (Hariharan season, heart rate was 151.3±10.8 beats min−1 at room temperature et al., 2016). (data not shown; N=7). We handled tenrecs while they were Tenrecs mated upon emergence from hibernation (∼September to hibernating at room temperature. Heart rate increased during November). Little is known of the reproductive biology of tenrecs handling to as high as 87 beats min−1 (71.2±5.15 beats min−1; other than that it seems to be plastic. In the lesser hedgehog tenrec, N=5 tenrecs) but quickly returned to values of 32.6 Echinops telfairi, gestation is reported to vary between 50 and ±2.04 beats min−1 within 94.4±35.7 s. Even with the reduced 79 days (Künzle et al., 2007). Gestation length in T. ecaudatus is heart rate, some tenrecs were able to ambulate (albeit these tenrecs suggested to be 58–64 days (Eisenberg, 1975). We have observed were ataxic). tenrecs giving birth as early as November and as late as February; To better understand this more or less torpid concept, we however, we are not familiar with any studies indicating how factors employed a RRT assay (Fig. 4). Our assay data confirmed our like lowered and variable Tb and/or use of facultative torpor might observations that hibernation in tenrecs represents a continuum, affect gestation length. Interestingly, pregnant tenrecs were found to with animals being most torpid in June during the austral winter display extremely variable Tb (as low as 13.5±0.5°C) with little (Fig. 4A). We found Tb to be a less than reliable indicator of apparent effect on their reproductive success (Fig. S6G; Nicoll, metabolic status. Tb partially predicts activity status, with warmer 1982). tenrecs being able to right themselves more quickly. However, not Common tenrec litter size may be enormous (as many as 32 all tenrecs were predictable, e.g. one tenrec with a Tb of 29.5°C babies), although litter sizes of 16–20 are more typical (Nicoll and experienced a 30 s RRT despite being housed at 12°C (Fig. S4). Racey, 1985). Our colony’s litter sizes were generally smaller, with Torpid tenrecs (Tb <20°C) experienced repeated cycles of a maximum of 19. Superfetation has been suggested in tenrecs relatively short, single-peak bursts followed by as much as 45 min (Poduschka, 1996). This ability to simultaneously support multiple of waning oxygen consumption (Fig. 3; Fig. S2). The ventilation litters at different developmental stages in the uterus is well pattern was reminiscent of insect discontinuous gas exchange documented in European hares (Roellig et al., 2011). In our limited (Lighton, 1998). This mechanism is proposed to allow efficient experience, one tenrec was determined to be pregnant, isolated from gas exchange in subterranean environments that become hypoxic further interactions with males, gave birth in isolation and was and hypercapnic. However, our experiments did not involve subsequently maintained in isolation for 40 days before giving birth exposure to hypoxia or hypercapnia. One possible function of to another litter. This occurrence suggests that, although rare, these brief bursts of metabolic activity may be that they serve the tenrecs may avail themselves of superfetation. A combination of same role as a canonical interbout arousal. For instance, one might large litter size and superfetation would suggest tremendous envisage periodic increases in heart rate as in Fig. 3 that may lead to reproductive output is available to these tenrecs. Journal of Experimental Biology

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Endothermy in modern placental mammals is widespread. References Although there are several tenable models for why endothermy may Barnes, B. M. (1989). Freeze avoidance in a mammal: body temperatures below 0°C in an Arctic hibernator. Science 244, 1593-1595. have evolved, all predict selective advantages to animals that maintain Bowen, W. D., Oftedal, O. T. and Boness, D. J. (1985). Birth to weaning in 4 days: warmer and more stable Tb (Lovegrove, 2016). Are tenrecs simply not remarkable growth in the hooded seal, Cystophora cristata. Can. J. Zool. 63, 2841-2846. able to maintain a warmer and more stable Tb?Wefoundfemale Buck, C. L. and Barnes, B. M. (2000). Effects of ambient temperature on metabolic tenrecs became increasingly endothermic and homeothermic on the rate, respiratory quotient, and torpor in an arctic hibernator. Am. J. Physiol. Regul. day of parturition (Fig. 6; Fig. S6). These data suggest that while Integr. Comp. Physiol. 279, R255-R262. Dausmann, K. H., Glos, J., Ganzhorn, J. U. and Heldmaier, G. (2004). tenrecs are normally very plastic and display highly variable Tb,they may experience periods wherein increased endothermy/homeothermy Physiology: hibernation in a tropical primate. Nature 429, 825-826. Dausmann, K. H., Glos, J., Ganzhorn, J. U. and Heldmaier, G. (2005). is possible. While T. ecaudatus display this increased endothermy/ Hibernation in the tropics: lessons from a primate. J. Comp. Physiol. B. 175, homeothermy post-parturition, work with other species of tenrec (E. 147-155. telfairi and Setifer setosus) suggests increased homeothermy while Dewasmes, G., Loos, N., Delanaud, S., Ramadan, W. and Dewasmes, D. (2003). Liver temperature during sleep. Sleep 26, 948-950. tenrecs are pregnant or across the breeding season in males (Oelkrug Eisenberg, J. F. (1975). Tenrecs and solenodons in captivity. Int. Zoo Yearb. 15, et al., 2013; Levesque and Lovegrove, 2014). 6-12. If increased endothermy/homeothermy were advantageous and Everson, K. M., Soarimalala, V., Goodman, S. M. and Olson, L. E. (2016). Multiple loci and complete taxonomic sampling resolve the phylogeny and given these tenrecs are capable of increased endothermy/ biogeographic history of tenrecs (mammalia: tenrecidae) and reveal higher homeothermy, then why not be more consistently homeothermic? speciation rates in Madagascar’s humid forests. Syst. Biol. 65, 890-909. The answer may lie in the advantages of being plastic. Many Garbutt, N. (1999). Mammals of Madagascar. Yale University Press. consider canonical mammalian hibernation as a means of passing the Gilmore, D. P., Da-Costa, C. P. and Duarte, D. P. F. (2000). An update on the physiology of two-and three-toed sloths. Braz. J. Med. Biol. Res. 33, 129-146. winter when resources are low. Tenrecs may simply be exploiting Hariharan, I. K., Wake, D. B. and Wake, M. H. (2016). Indeterminate growth: could it hibernation-like strategies in their active-season physiology. Varying represent the ancestral condition? Cold Spring Harb. Perspect. Biol. 8, a019174. Jani, A., Martin, S. L., Jain, S., Keys, D. and Edelstein, C. L. (2013). Renal metabolism and Tb would presumably allow for the conservation of adaptation during hibernation. Am. J. Physiol. Renal Physiol. 305, F1521. energetic stores. One can then flip this argument around and ask why Johansen, K. (1962). Responses to heat and cold in lower mammals. a placental mammal would be less plastic. That answer may lie in the Int. J. Biometeorol. 6, 3-28. limits to plasticity. Despite being considered a hypervariable Karpovich, S. A., Tøien, Ø., Buck, C. L. and Barnes, B. M. (2009). Energetics of environment, much of Madagascar is rather warm (Ohba et al., arousal episodes in hibernating arctic ground squirrels. J. Comp. Physiol. B 179, 691-700. 2016). In fact, Madagascar’s climate is thought to have changed very Kayser, C. (1961). The Physiology of Natural Hibernation. Oxford, New York: little since tenrecs first colonized the island 30–56 MYA (Everson Pergamon Press. et al., 2016; Ohba et al., 2016). We found tenrecs appear highly Künzle, H., Nautrup, C. P. and Schwarzenberger, F. (2007). High inter-individual variation in the gestation length of the hedgehog tenrec, Echinops telfairi stressed when Ta or Tb is <8°C or >34°C as cold animals may cease (Afrotheria). Anim. Reprod. Sci. 97, 364-374. ventilating and hot animals pant and breathe irregularly (Kayser, Levesque, D. L. and Lovegrove, B. G. (2014). Increased homeothermy during 1961; Oelkrug et al., 2013; Scholl, 1974; M.D.T., L.S., B.B. and reproduction in a basal placental mammal. J. Exp. Biol. 217, 1535-1542. F.v.B., personal observations). In the wild, the likelihood of tenrecs Levesque, D. L., Lobban, K. D. and Lovegrove, B. G. (2014). 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The evolution of endothermy in Cenozoic mammals: a Interestingly, the ranges of extant mammals like monotremes, plesiomorphic-apomorphic continuum. Biol. Rev. 87, 128-162. marsupials, afrotherians and xenarthrans favor more moderate Lovegrove, B. G. (2016). A phenology of the evolution of endothermy in birds and climates (Johansen, 1962). mammals. Biol. Rev. 92, 1213-1240. Lovegrove, B. G., Lobban, K. D. and Levesque, D. L. (2014). Mammal survival at the Cretaceous– Palaeogene boundary: metabolic homeostasis in prolonged Acknowledgements tropical hibernation in tenrecs. Proc. R. Soc. B 281, 20141304. We thank Barry Lovegrove and Owen Griffith for assistance acquiring our tenrec Mcmanus, J. J. (1969). Temperature regulation in the opossum, Didelphis colony. We also thank the numerous undergraduates and volunteers from the van marsupialis virginiana. J. Mammal. 50, 550-558. Breukelen laboratory for assistance with animal husbandry and maintenance. Mzilikazi, N. and Lovegrove, B. G. (2004). Daily torpor in free-ranging rock elephant shrews, Elephantulus myurus: a year-long study. Physiol. Biochem. Competing interests Zool. 77, 285-296. The authors declare no competing or financial interests. Mzilikazi, N. and Lovegrove, B. G. (2005). Daily torpor during the active phase in free-ranging rock elephant shrews (Elephantulus myurus). J. Zool. 267, 103-111. Author contributions Nicoll, M. E. (1982). Reproductive ecology of Tenrec ecaudatus (Insectivora: Conceptualization: M.D.T., F.v.B.; Methodology: M.D.T., J.R., J.R.B.L., F.v.B.; Tenrecidae) in the Seychelles. Doctoral dissertation, University of Aberdeen. Software: J.R., J.R.B.L.; Formal analysis: M.D.T.; Investigation: M.D.T., L.S., B.B., Nicoll, M. E. and Racey, P. A. (1985). Follicular development, ovulation, fertilization A.K., A.J.M., T.R., A.R., C.F.R., D.S., J.F.S., C.S.R., E.S., J.R., J.R.B.L.; Data and fetal development in tenrecs (Tenrec ecaudatus). J. Reprod. Fertil. 74, 47-55. Oelkrug, R., Meyer, C. W., Heldmaier, G. and Mzilikazi, N. (2012). Seasonal curation: M.D.T., D.S., J.F.S., C.S.R.; Writing - original draft: M.D.T., F.v.B.; Writing - changes in thermogenesis of a free-ranging afrotherian small mammal, the review & editing: M.D.T., F.v.B.; Visualization: M.D.T., F.v.B.; Supervision: M.D.T., western rock elephant shrew (Elephantulus rupestris). J. Comp. Physiol. B 182, F.v.B.; Project administration: M.D.T., F.v.B.; Funding acquisition: F.v.B. 715-727. Oelkrug, R., Goetze, N., Exner, C., Lee, Y., Ganjam, G. K., Kutschke, M., Müller, Funding S., Stöhr, S., Tschöp, M. H., Crichton, P. G. et al. (2013). Brown fat in a This study was funded by National Science Foundation grants IOB 0448396 and protoendothermic mammal fuels eutherian evolution. Nat. Commun. 4, (2140). IOS 1655091. Ohba, M., Samonds, K. E., Lafleur, M., Ali, J. R. and Godfrey, L. R. (2016). Madagascar’s climate at the K/P boundary and its impact on the island’s biotic Supplementary information suite. Palaeogeogr. Palaeoclimatol. Palaeoecol. 441, 688-695. Supplementary information available online at O’Leary, M. A., Bloch, J. I., Flynn, J. J., Gaudin, T. J., Giallombardo, A., http://jeb.biologists.org/lookup/doi/10.1242/jeb.185900.supplemental Giannini, N. P., Goldberg, S. L., Kraatz, B. P., Luo, Z.-X., Meng, J. et al. (2013). Journal of Experimental Biology

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